DNA, RNA, immunoglobulins and proteins are classes of polymeric biomolecules (“biopolymers”) of particular importance in modern biochemical and molecular biological methods and processes. Specifically, biopolymers play critical roles in various subcellular processes including the preservation and transmission of genetic information, the production of proteins, and the formation of enzymes.
Because of the importance of these biopolymers in various biological processes, a wide variety of techniques have been developed to physically bind these classes of molecules in order to achieve their immobilization, purification, concentration, archival storage, etc., i.e., in order to manipulate such molecules. For example, various column methods have been developed to bind a biopolymer to a matrix with affinity for that biopolymer, thereby allowing for its immobilization, its separation from contaminating cellular components, its concentration, etc. See, e.g., U.S. Pat. No. 5,652,141 for the purification of nucleic acids (i.e., DNA and RNA). Similarly, various bulk separation methods have been developed for the immobilization, separation, or concentration of biopolymers, e.g., the use of magnetic beads comprising a magnetic or paramagnetic particle and an attached ion exchange material capable of binding the biomolecule. See, e.g., U.S. Pat. No. 6,718,742.
Biopolymer immobilization, separation, concentration or purification is employed across a wide range of commercial applications, including, for example, forensics, pharmaceutical research and development, medical diagnostics and therapeutics, environmental analysis, such as water purification or water quality monitoring, nucleic acid purification, proteomics, and field collection of biological samples. Thus, a great need exists for efficient, simplified processing of clinical, environmental and forensic samples; especially those containing nanogram amounts of nucleic acid or protein.
There are generally three classes of current technology for nucleic acid extraction and purification from fluidized cellular or tissue suspensions: a) Organic Precipitation; b) Adsorption to Glass Column or Fiber Supports; and c) Adsorption to Magnetic Beads.
Organic precipitation is generally the method of choice for extraction of samples larger than about 1 ml, because it scales to large size well. It is based on the semi-selective precipitation of high molecular weight DNA and RNA, induced by organic solvents and chaotropes, followed by harvesting of the precipitate by high speed centrifugation. The primary precipitate, which is only semi-pure, is then dissolved and additionally purified by protease treatment and ordinarily, a second round of precipitation at −20° C., in an organic solvent.
One disadvantage of the precipitation method lies in the complexity and the need to have access to cryogenics and a high speed preparative centrifuge to affect precipitation. The methods are generally unsuited to routine automation. For this reason, the precipitation method is currently used mainly in low volume applications, as in a bench scale biochemistry laboratory, and especially for large volume samples (in excess of 5 mL) such as 8 mL blood samples or plant or animal tissue extracts.
Adsorption onto a glass column or fiber supports is based upon adsorption of nucleic acid to a column of porous glass beads or to filters made from glass fibers. It is performed by lysing a fluidized sample as would be done in the precipitation method, but instead of precipitation of the nucleic acid, the nucleic acid is induced to adsorb onto a treated glass surface: generally as a result of adding salts, chaotrope or alcohol to the fluid phase, to make the nucleic acid less soluble. Once adsorbed to the solid support (column or fiber), contaminants are flushed away by washing of the solid support with an appropriate wash solution, usually containing alcohol to retain tight nucleic acid adsorption. Finally, nucleic acid is released from the support by addition of an alcohol-free buffer, thereby eluting the nucleic acid into a small volume for subsequent genetic analysis.
This method is widely used as the basis for commercial products. One advantage of this method is that it is relatively simple, requires somewhat less laboratory infrastructure than the biochemical precipitation method and because it is relatively easy to automate, in a 96 well microplate format. Another disadvantage of the method is that, as for precipitation, it requires rather precise control of the lysis, washing and elution steps in order to obtain high yields and high purity. Moreover, as for the precipitation methods, it requires access to relatively sophisticated biochemical lab equipment: centrifuges, pressure filtration devices, etc. Yet another disadvantage is that it does not scale well to large sample size. Thus, the glass adsorption method is superior to the precipitation methods and is very well suited for samples less than about 1 ml, especially when standardized automation is a requirement for high throughput applications. A final disadvantage is one of cost. Since the adsorption methods must be used in the context of a column-based chromatographic separation or a filtration based separation, they are provided commercially as an integrated plastic device (spin columns, filter-bottom microplates, etc) which adds significant consumable cost to the overall process.
Adsorption onto magnetic beads entails using plastic coated micron-sized beads (that have a density near to one, and a size range from 1 to 50 microns) with iron, or an equivalent diamagnetic substance at their center. Because these beads have a density near to that of water, they cannot be easily manipulated by centrifugation. However, because they have an iron core, they can be drawn to the bottom or the side of a vessel by a magnet. For nucleic acid purification, such beads are coated with a neutral or cationic coating and, similar to the glass bead technology, nucleic acid is purified by adsorption to the bead surface: but followed by bead isolation in the presence of a nearby magnet, thereby drawing bead-bound nucleic acid to the bottom of a preparative tube.
In contrast to the precipitation or glass bead or filtration technology, an advantage of the magnetic bead technology is that it requires very little specialized laboratory equipment (other than the magnet) and because it is readily automated. It is considered superior to both the glass bead and the precipitation method in that the final nucleic acid product can be isolated as a very small volume pellet, which, subsequent to addition of a small amount of release buffer, can be harvested as a microliter-sized product which in volume terms, can be 1/10 that provided by release from glass columns or glass filters.
Disadvantages of the magnetic bead technology are as follows. Because it is not practical to apply a magnetic field which will “pull” beads of this kind more than about 1 cm, this technology does not scale well to the isolation of nucleic acid from samples larger than about 1 mL. Since magnetic attraction is proportional to the mass of the diamagnetic material, these non-porous magnetic beads are limited in to a size ranging from 1 to 50 microns (because of the magnetic core) which is why there is a severe limitation as to the surface area available per initial unit sample volume. Moreover, the beads themselves are relatively complicated to manufacture (they have a magnetic core and a surface coating) and are therefore costly. Thus, as is the case for the glass adsorption technology, the magnetic bead technology is relatively costly and best suited for small volume sample processing.
Although each of these conventional techniques are capable of manipulating biopolymers, they suffer various difficulties in terms of cost of operation, complexity of apparatus required, amount of biopolymer obtained, concentration of biopolymer obtained, etc. In light of the importance of biopolymers to modem biological research such as the development of new therapeutic treatments, drugs, etc., there is thus a need for alternate methods for manipulating such biopolymers that address these various deficiencies in current techniques.
In contrast to the conventional nucleic acid extraction and purification methodologies, the inventive method employs sub-micron or nanoparticles that are readily available in kilogram quantities. One of the preferred nanoparticle materials, kaolin, is commercially available at high purity and at very low cost, since these materials are already used industrially as fillers and bonding agents. The coupling process that we have devised can prepare surface modified particles (e.g., epoxy-silane coated kaolin) batch-wise in kilogram quantities, under conditions where the production cost for the final product will be dominated by QC-QA and packaging. Thus, the overall cost of producing the chromatographically-useful nanoparticles as a technology will be similar to that of the low-cost precipitation methods and approximately 1/10 that of the glass column or glass filter or magnetic bead technologies.
Like the glass column/glass filter technology and the magnetic bead technology, the kaolin nanoparticle technology can be readily automated, in a 96 well or 384 well format, mediated by very low speed particle isolation by gravity or low speed plate-wise centrifugation. Like the magnetic bead technology, chromatography with the nanoparticles of this invention can be performed with little to no specialized equipment: only an inexpensive low speed blood centrifuge and a pipetter. However, unlike either the glass bead or magnetic bead technology, the extraction process with nanoparticles of this invention can be scaled from the microliter scale to the liter scale effortlessly, since nucleic acid isolation is based on the application of a moderate centrifugal force allowing samples to be purified, alternatively in an “Eppendorf” tube or a blood “stick tube” or even a 250 ml blood bottle, if necessary. Also, the cost of material and manufacture is very low, and the material can be used without the need for embedding it in an expensive custom chromatography or filtration part (or coupled to the use of expensive magnets). The overall cost to the customer can be much lower than the industry standard, with equal or greater quality.
The present invention provides a unique chromatographic matrix based on highly purified, nearly mono-dispersed, ceramic nanoparticles. These nanoparticles form stable colloidal suspensions in aqueous solution, but are dense enough that they readily sediment and form a compact pellet in response to standard low speed bench top centrifugation. Since the particles are sub-micron in diameter, they display a large surface area to volume ratio. For example, a milligram of kaolin nanoparticle of 200 nm dimension, suspended in one milliliter of aqueous solution displays a total surface area in the range of 200 cm2 that represents approximately 1012 dispersed nanoparticles The inter-particle spacing between this number of particles in a milliliter is a about a micron, a distance that is less than the distance a 10,000 base pair long DNA molecule or a 1,000,000 D protein would travel by passive diffusion in about one minute. Thus, even at a very low particle-mass density, and in the absence of mixing or convective flow, a 0.1% suspension of nanoparticles are at a concentration such that a targeted biomolecule is never more than “a minute away” from binding to the nanoparticle. This suggests that, independent of sample concentration, batch chromatography with nanoparticles will be complete within minutes. In addition, due to the small size of these particles, this 0.1% suspension of kaolin nanoparticles will sediment to a pellet volume of only about one microliter. The expansive surface area of these ceramic nanoparticles has very useful characteristics as the basis for chromatography: an enormous binding capacity per unit mass and the ability to be modified via well-known surface chemistry.
For batch chromatography, the outer surface area is important because it defines the mass and the volume of sample that can be processed at one time. Since the surface area per unit mass increases as 1/diameter, to make the comparison to the inventive kaolin nanoparticles, having a surface to mass of 200 cm2 per milligram, for a smooth, non-porous particle of a 30 micron size beads or glass (same approximate density as kaolin and a surface area to mass ratio of 2 cm2/mg), would require 100 milligrams of beads to match the surface area of 1 mg of the kaolin nanoparticles. Assuming that each kind of matrix occupies about the same space per unit mass as a pellet, the surface area presented by 1 μL of a 200 nm-nanoparticle pellet is equivalent to that of a 100 μL pellet for the standard 30-micron bead. Thus, in this very realistic example, a biologically relevant sample would have been concentrated 1000-fold via nanoparticles, but only 10-fold by the 30 uM beads. Alternatively, in terms of binding capacity, a 100 μL pellet would have the same total binding capacity as 1 uL of the nanoparticles.
Rapid development of a broad-ranging nano-chromatography platform requires the ability to chemically modify the surface of this new ceramic matrix with biomolecule-specific ligands. For ceramic nanoparticles, surface chemistry is well studied and has already been optimized for related applications in the plastics and polymers industry. In the present invention these well-known ceramic surface chemistries are paired with biochemical chromatography, to develop a flexible repertoire of surface coatings for DNA, RNA, immunoglobulin and protein applications: all based on the same underlying ceramic nanoparticle matrix.
With regard to the immobilization and storage of biopolymers, the inventive nanoparticles can be used for the storage and retrieval of nucleic acid and proteins in the solid state. It is well known in the art that immobilization of biomolecules on a surface stabilizes the molecule against changes due to environmental or enzymatic degradation. There are several other known methods of nucleic acid storage, commercial and noncommercial, namely: a) cryogenics; b) unassisted air or freeze drying; and c) drying on treated paper filters. Since these nanoparticles present such a large surface area, the expectation is that the immobilization or adsorption of nucleic acids or proteins on specially modified surfaces of these nanoparticles would be a means to store these biomolecules for later analysis.